Enzymatic Assay of Fumarase

1. OBJECTIVE
To standardize a procedure for the enzymatic assay of Fumarase in Analytical Services at Sigma-Aldrich - St. Louis.

2. SCOPE
The scope of this procedure is for all products that have a specification for Fumarase activity including, but not limited to, Sigma-Aldrich Product Number F1757.

3. DEFINITIONS

3.1 Purified Water = water from a deionizing system, resistivity > or = 18MΩ•cm @ 25ºC

3.2 Unit Definition - One unit will convert 1.0 μmole of L-Malate to Fumarate per minute at pH 7.6 at 25ºC.

4. DISCUSSION
L-Malate    Fumarase   > Fumarate + H2O

5. RESPONSIBILITIES
It is the responsibility of trained Analytical Services laboratory personnel to follow this procedure as written.

6. SAFETY
Refer to Material Safety Data Sheets (MSDS) for hazards and appropriate handling precautions.

7. PROCEDURE

7.1 CONDITIONS:
T = 25°C, pH = 7.6, A240nm, Light path = 1 cm

7.2 METHOD:
Continuous Spectrophotometric Rate Determination

7.3 REAGENTS:

7.3.1    100mM Potassium Phosphate Buffer, pH 7.6 at 25ºC (Buffer)
             Prepare a 14 mg/ml solution in purified water using Potassium Phosphate, such as Sigma-Aldrich Product Number P5379. Adjust to pH 7.6 at 25ºC with 1 N KOH.

7.3.2    50mM L-Malic Acid (Malate)
             Prepare a 7 mg/ml solution in Reagent 7.3.1 (Buffer) using L-Malic Acid, such as Sigma-Aldrich Product Number M1000. Adjust to pH 7.6 at 25ºC with KOH.

7.3.3    0.1% Bovine Serum Albumin (BSA)
            Prepare a 1 mg/ml solution in purified water using Bovine Serum Albumin, such as Sigma-Aldrich Product Number A6003.

7.3.4    Fumarase Enzyme Solution (Enzyme)
            Immediately before use, prepare a solution containing 1 unit/mL of Fumarase in cold Reagent 7.3.3 (BSA). The enzyme solution MUST BE CLEAR AND COLORLESS before addition to the reaction mixture. Any cloudiness indicates the enzyme is not completely solubilized.

7.4 TEST METHOD

7.4.1    Pipet (in milliliters) the following reagents into suitable quartz cuvettes:

  Blank Test-1 Test-2 Test-3
Reagent 7.3.2 (Malate) 2.90 2.90 2.90 2.90
Reagent 7.3.3 (BSA) 0.10 ---- ---- ----

7.4.2    Mix by inversion and equilibrate to 25ºC. Monitor the A240nm until constant, using a suitably thermostatted spectrophotometer. Then add:

Reagent 7.3.4 (Enzyme) ---- 0.10 0.07 0.05

7.4.3    Immediately mix by inversion and record the increase in A240nm for approximately 10 minutes. Obtain the ΔA240nm/minute using the maximum linear rate.

7.5 CALCULATIONS

7.5.1 Units/ml = (ΔA240nm/minute test - ΔA240nm/minute blank)(TV)(df)
(2.44)(SV)

where:
    df = Dilution Factor
    TV = Volume (in milliliters) of assay, differs for Test 1,2,3
    2.44 = millimolar extinction coefficient of Fumarate at 240nm
    SV = Volume (in milliliters) of enzyme used, differs for Test 1,2,3


  Units/mg protein = Units/mL enzyme
mg protein/mL enzyme

7.6 FINAL ASSAY CONCENTRATION :
In a 3 mL reaction mix, the final concentrations are 96.7 mM potassium phosphate, 48.3 mM L-malic acid and 0.1 unit fumarase.

8. REFERENCES & ATTACHMENTS

8.1 Bergmeyer, H.U., Gawehn, K., and Grassi, M. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed) Volume I, 452, Academic Press, Inc., New York, NY.

8.2 Replaces SPMALA01 (v1) for Analytical Services use.

9. APPROVAL

Review, approvals and signatures for this document will be generated electronically using the EDMS. Print a “For Use” copy if hardcopy with signature verification is required.

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